Development of multi functionalized polymeric carriers for delivery of anticancer drug combinations

DEVELOPMENT OF MULTI-FUNCTIONALIZED POLYMERIC CARRIERS FOR DELIVERY OF ANTICANCER DRUG COMBINATIONS DUONG HOANG HANH PHUOC NATIONAL UNIVERSITY OF SINGAPORE 2013 DEVELOPMENT OF MULTI-FUNCTIONALIZED CARRIERS FOR DELIVERY OF ANTICANCER DRUG COMBINATIONS DUONG HOANG HANH PHUOC (B. Eng., HOCHIMINH UNIVERSITY OF TECHNOLOGY, VIETNAM) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Duong Hoang Hanh Phuoc 29 November 2013 I ACKNOWLEDGEMENTS First of all, I would like to express my deepest and most sincere gratitude to my supervisor, Professor Yung Lin Yue Lanry, for his endless help, support, guidance, and patience. Without his extremely generous help and support, it would have been impossible for me to accomplish my PhD study. I deeply appreciate him for giving me not only a lot of opportunities to learn but also freedom to try and explore new ideas. I am grateful to his advice, encouragement and care not only in research works but also in personal matters. I am privileged to have him, not just as a great and thoughtful supervisor, but as a good friend as well. I would like to thank all friends and fellow graduate students in Prof. Yung’s and Prof. Tong’s lab, past and present, especially Ms Tan Weiling, Dr Deny Hartono, Miss Fong Kah Ee, Dr Zhao Shuang, Dr Luo Jingnan for their unconditional help and encouragement. I would like to convey my thanks to all lab technologists and friends from Chemical & Biomolecular Engineering Department of NUS whom I had worked closely with during my PhD study. I would like to express my thanks to Mdm Li Xiang for all her help, care and positive encouragement. I would like to acknowledge National University of Singapore for giving me a research scholarship to pursue my PhD study. II Last but not least, I would like to express my most sincere appreciation to my family members for all their constant love, encouragement and support. My gratitude also goes to all other friends that had supported me in many ways during my PhD study. III TABLE OF CONTENTS DECLARATION . I ACKNOWLEDGEMENTS II TABLE OF CONTENTS IV SUMMARY IX LIST OF TABLES . XII LIST OF FIGURES . XIV CHAPTER 1. Introduction 1.1 Background . 1.1.1 Cancer 1.1.2 Limitation of traditional chemotherapeutic technology for cancer treatments 1.1.3 Requirements for an ideal drug delivery system 1.2 Hypotheses 1.3 Objectives and scope of the study . CHAPTER 2. Literature Review 10 2.1 Cancer treatment . 10 2.2 Traditional cancer chemotherapy technology . 11 2.3 Drug delivery technology . 13 2.4 Common carriers for anticancer drug delivery . 16 2.4.1 Liposomes 16 2.4.2 Polymer-drug conjugates . 18 2.4.3 Polymeric nanoparticles (NPs) 20 2.4.4 Polymeric micelles . 22 2.5 Overview of current drug delivery strategies 28 2.5.1 Passive delivery . 29 2.5.2 Active delivery by targeting to cancer cells . 30 2.5.3 Active delivery by targeting to endothelial cells . 32 2.5.4 Cell-penetrating peptides . 33 IV 2.6 Combination chemotherapy 36 2.6.1 Overview of combination chemotherapy . 36 2.6.2 Principle of drug selection in the combination 37 2.6.3 Some commonly used anticancer drugs and their combinations . 39 2.6.4 Determination of combined chemotherapeutic effect 41 CHAPTER 3. Surface modification of polymeric micelle particles for enhancement of cancer targeting and penetrating ability 44 3.1 Introduction . 44 3.2 Experimental section . 47 3.2.1 Materials 47 3.2.2 Synthesis of PLGA-PEG 48 3.2.3 Synthesis of PLGA-PEG-FOL . 48 3.2.4 Synthesis of PLGA-PEG-TAT 49 3.2.5 Characterization of polymers . 50 3.2.6 Critical micelle concentration (CMC) . 51 3.2.7 Preparation and characterization of doxorubicin loaded polymeric micelles 51 3.2.8 In vitro release of doxorubicin (DOX) . 52 3.2.9 Preparation of Coumarin 6-loaded micelles 53 3.2.10 In vitro cellular uptake . 53 3.2.11 In vitro cytotoxicity of DOX-loaded micelles . 54 3.3 Results and discussion 54 3.3.1 Characterization of PLGA-PEG 54 3.3.2 Characterization of PLGA-PEG-FOL 56 3.3.3 Characterization of PLGA-PEG-TAT . 56 3.3.4 Critical micelle concentration (CMC) . 57 3.3.5 Particle size, zeta potential . 59 3.3.6 In vitro drug release and drug loading . 60 3.3.7 Cytotoxicity of DOX- loaded micelles 61 3.3.8 Cellular uptake . 67 3.4 Conclusions . 68 CHAPTER 4. Synergistic co-delivery of doxorubicin and paclitaxel using multifunctionalized micelles for cancer treatment 70 V 4.1 Introduction . 70 4.2 Experimental section . 72 4.2.1 Materials 72 4.2.2 Preparation and characterization of doxorubicin (DOX) and paclitaxel (PTX) loaded polymeric micelles 73 4.2.3 In vitro release study 75 4.2.4 In vitro cytotoxicity study 76 4.2.6 Determination of combination effects . 76 4.3 Results and discussion 77 4.3.1 In vitro cytotoxicity interaction between free doxorubicin (DOX) and free paclitaxel (PTX) 77 4.3.2 Size and zeta potential characterization of drug-loaded polymeric micelles . 81 4.3.3 In vitro drug release and drug loading of singe drug-loaded micelles . 82 4.3.4 In vitro drug release and drug loading of dual drug-loaded micelles 84 4.3.5 Cytotoxicity enhancement of drug-loaded micelles with the addition of TAT on the micelle surface . 86 4.3.6 Synergistic effect of the co-delivery of DOX- loaded micelles and PTX-loaded micelles . 90 4.3.7 Synergistic effect of dual drugs-loaded micelles and the surface modifications . 92 4.4 Conclusions . 93 CHAPTER 5. Dual-functionalized micellar system for synergistic delivery of hormone therapeutic and chemotherapeutic agents for breast cancer treatment 95 5.1 Introduction . 95 5.2 Experimental section . 100 5.2.1 Materials 100 5.2.2 Preparation and characterization of PTX and TAM loaded polymeric micelles . 100 5.2.3 In vitro release study 101 5.2.4 In vitro cellular uptake . 102 5.2.4 In vitro cytotoxicity study 102 5.2.5 Median-effect analysis . 103 5.3 Results and discussion 103 VI 5.3.1 In vitro cytotoxicity interaction between free tamoxifen (TAM) and free paclitaxel (PTX) 103 5.3.2 Characterization of drug-loaded polymeric micelles . 107 5.3.3 Enhancement of drug-loaded micelles with the surface modification using combined TAT and FOL . 109 5.3.4 Synergistic effect of the co-delivery of TAM-TAT/FOL micelles and PTXTAT/FOL micelles 113 5.3.5 Synergistic effect of dual drugs-loaded micelles (TAM/PTX-TAT/FOL micelles) 114 5.4 Conclusions . 117 CHAPTER 6. Targeting delivery of a synergistic combination of doxorubicin and cisplatin with polymer-drug complex micellar systems . 119 6.1 Introduction . 119 6.2 Experimental section . 122 6.2.1 Materials 122 6.2.2 Synthesis and characterization of polymers . 123 6.2.3 Preparation and characterization of cisplatin (CDDP) and doxorubicin (DOX) micelles . 126 6.2.4 In vitro release study 127 6.2.5 In vitro cytotoxicity study 127 6.3 Results and discussion 128 6.3.1 Characterization of polymers . 128 6.3.2 In vitro cytotoxicity interaction between free cisplatin (CDDP) and free doxorubicin (DOX) . 130 6.3.3 Characterization of drug-loaded micelles 133 6.3.4 In vitro drug release study 134 6.3.5 Cytotoxicity enhancement of drug-loaded micelles with the addition of FOL on the micelle surface . 136 6.3.6 Synergistic effect of the co-delivery of CDDP-loaded micelles and DOXloaded micelles 139 6.3.7 Synergistic effect of dual drugs-loaded micelles . 141 6.4 Conclusions . 142 CHAPTER 7. 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Ahmed, Polymersomes, 2006. p. 323-341. 173 [...]... inorganic drug delivery systems for cancer diagnosis and therapy [39] 14 Figure 2.4 Schematic of delivery mechanism of drug- loaded carriers to tumor cells [42, 45] 15 Figure 2.5 Schematic of drug- loaded liposome formation 16 Figure 2.6 Schematic of drug delivery system using polymer -drug conjugate system [70] 19 Figure 2.7 Schematic of polymer -drug. .. combinations of anticancer drugs are co-encapsulated into the micelles (4) Polymeric micelles can also be used as carriers for hydrophilic drug delivery if the drugs and polymers exhibit specific chemical interactions 1.3 Objectives and scope of the study The objectives of this thesis are to investigate and demonstrate polymeric micellar drug delivery systems for cancer therapy to address the limitations of. .. 21 Figure 2.8 Schematic of preparation of physical drug- loaded polymeric micelles 23 Figure 2.9 Schematic of BIND-014, a docetaxel (DTXL)-loaded micelle system with small-molecule (ACUPA) targeting ligands 27 Figure 2.10 Schematic of multifunctional polymeric carriers for active drug delivery [24] 29 Figure 2.11 Schematic of a passive targeted drug delivery system [31] ... different pair of drugs have been investigated XI LIST OF TABLES Table 2.1 Anticancer therapeutic and their mechanism of action [24] 12 Table 2.2 Sample of some liposome-based drugs for cancer chemotherapy 17 Table 2.3 Sample of polymer -drug conjugates 20 Table 2.4 Nanoparticle-based drugs for cancer chemotherapy [31, 86] 22 Table 2.5 Drug- loaded polymeric micellar formulations ... clearance, and fast development of multipledrug resistance (MRD) Figure 2.2 Schematic representation of the delivery mechanism of small-molecule drugs to tumors [31] 2.3 Drug delivery technology To overcome the limitations of the typical chemotherapy, drug delivery systems have been developed to generate new therapeutic systems with better treatment efficacy and lower side effects Numerous drug delivery systems... resultant drug- loaded micelles exhibit suitable properties for drug delivery (2) Multi- modification of micelles with different moieties which are specially used for drug delivery systems can increase the treatment efficacy of the resultant micelles compared to that of single-moiety modified one (3) It has been further hypothesized that the drug delivery system is more effective when synergistic combinations. .. advantages of (1) synergistic effect of combined drugs, (2) polymeric carrier for drug delivery with sustained release and biocompatibility properties, (3) carrier modifications with targeting moiety to enhance the delivery selectivity and/or with penetrating peptide to enhance the uptake The comparisons between the co -delivery of two single drug- loaded carrier systems and the delivery of dual-drugs-loaded... first objective of this work is to develop an effective system for anticancer drug delivery The system has been developed for physically encapsulating of hydrophobic drugs because most of anticancer drugs are hydrophobic in nature Self-assembled polymeric micelles based on biodegradable amphiphilic copolymer poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)(PLGAPEG) have been multi- functionalized. .. 2.3 Schematic of organic and inorganic drug delivery systems for cancer diagnosis and therapy [39] The existing challenges of drug delivery system are to design suitable carriers that can efficiently encapsulate anticancer drugs, overcome drug- resistance, and increase selectivity of drugs towards cancer cells while eliminating their toxicity to normal tissues To efficiently encapsulate drugs into a... dysfunctional lymphatic drainage of tumors [4144] The uptake of a drug delivery system can also be enhanced by decorating the carrier with specific ligands In addition other important properties of carriers have also been considered for designing a drug delivery system including biocompatibility and low toxicity Figure 2.4 Schematic of delivery mechanism of drug- loaded carriers to tumor cells [42, 45] . DEVELOPMENT OF MULTI- FUNCTIONALIZED POLYMERIC CARRIERS FOR DELIVERY OF ANTICANCER DRUG COMBINATIONS DUONG HOANG HANH PHUOC NATIONAL UNIVERSITY OF SINGAPORE. SINGAPORE 2013 DEVELOPMENT OF MULTI- FUNCTIONALIZED CARRIERS FOR DELIVERY OF ANTICANCER DRUG COMBINATIONS DUONG HOANG HANH PHUOC (B. Eng., HOCHIMINH UNIVERSITY OF TECHNOLOGY, VIETNAM). characterization of drug- loaded polymeric micelles 81 4.3.3 In vitro drug release and drug loading of singe drug- loaded micelles 82 4.3.4 In vitro drug release and drug loading of dual drug- loaded